EPO is a glycoprotein hormone with a molecular weight of about 34 kD. EPO in the plasma is composed of 165 amino acids with a high degree of glycosylation, wherein the major component of glycosyl is sialic acid. Based on carbohydrate content, naturally occurring EPO is divided into two types, namely α and β-type, wherein α-type contains 34% carbohydrate and β-type contains 26% carbohydrate. These two types have the same biological characteristics, antigenicity and clinical effect. The human EPO gene is located on chromosome 7 long-Area 22. Its cDNA was successfully cloned in 1985, and recombinant human EPO (rHuEPO) has been largely produced using gene recombinant technology and widely used in the clinic. EPO has been biosynthesized using recombinant DNA technology (Egrie, J. C., Strickland, T W, Lane, J. etc. (986), Immunobiology, 72: 213-224). The biosynthesized EPO is the expression product of cloned human EPO gene in Chinese hamster ovary cells (CHO cells). Naturally occurring human EPO is firstly translated into polypeptide chains containing 166 amino acids with position 166 being arginine. In the post-translational modifications, arginine on position 166 is degraded using hydroxyl peptidase. The molecular weight of human EPO without glycosyl is 18236 Da. In the intact EPO molecule, glycosylation accounts for about 40% of the total molecular weight (J. Biol. Chem., 262: 12059).
EPO is the first cytokine applied in clinic and by far the best known hemoglobin-increasing drug with unique and safe effects. It has certain therapeutic effects on renal anemia, aplastic anemia, multiple myeloma, and paroxysmal nocturnal hematuria. In addition, the application of EPO can reduce the requirement of blood transfusion in surgery, and to some extent cure anemia caused by malignant tumor, chemotherapy and rheumatoid arthritis. Since EPO is primarily generated by renal tubular endothelial cells, anemia caused by renal disease is the first indication of EPO. The curative efficacy of EPO on renal anemia is almost 100%, but EPO does not improve renal function. Treatment with EPO is safe, effective and suitable for long-term treatment. In addition, it addresses the problem of shortage of blood supply. On the global market for biotechnology drugs in 2006, EPO recombinant drugs accounted for 11.9 billion U.S. dollars. There exists a huge market capacity.
As early as 1989, recombinant human EPO (EPOGEN®) was approved by U.S. FDA for the treatment of renal anemia, but it entered Chinese market only in 1992. The annual morbidity of chronic nephritis is about 0.25% in China, of which a considerable proportion of patients will eventually develop renal failure. The number of patients with renal anemia is about 500-600 thousands each year. According to conservative estimates of consumption of the drug in the renal anemia, together with consumption in other cancer-related anemia, the domestic market capacity is about 1.2-1.6 billion CNY or even more (calculated with the current price being 30-40 CNY/dose, the average weight of patients being 50 Kg). From the late 1990s, EPO has been ranked among the best-selling drugs in China's major cities. The cost of EPO is 62.13 million CNY in sample hospitals of major cities all over China in 2003, ranking number 56. The cost of EPO in sample hospitals of major cities all over China increased to 80.49 million CNY in 2004, with an annual increase of 30%.
As an endogenous hormone acting on the marrow hematopoietic cells to promote proliferation, differentiation and ultimate mature of erythroid progenitor cells, EPO plays an important role in regulating oxygen status of the body. In the early embryo stage, EPO is generated by the liver, then the production site of EPO gradually shifts to the kidney. EPO is mainly secreted by renal tubular interstitial cells after birth.
During induction of red progenitor cell differentiation by EPO, the globulin synthesis is induced, which allows cells to recruit more hemoglobin with functions of heme synthesis. The hemoglobin can combine with oxygen in mature red blood cells. Therefore, red blood cell and hemoglobin play an important role in supplying oxygen to the body. This process is initiated by the interaction between EPO and surface receptors of red progenitor cells.
When the body is in a healthy state, the tissue may obtain enough oxygen from already existing red blood cells. At this time the body's EPO concentration is very low. This low but normal concentration of EPO is sufficient to stimulate the generation of red blood cells, which is normally lost during aging.
When the level of oxygen transported by red blood cells in the circulation system is reduced and hypoxia appears, the amount of EPO in the body will increase. The body's hypoxic state may be caused by the following reasons: excessive exposure to radiation, reduced oxygen intake due to high latitude or long term coma, various types of anemia and so on. As a response to hypoxic stress of the body, a higher level of EPO can stimulate the differentiation of red progenitor cell to enhance its ability to produce red blood cells. When the number of red blood cells in the body exceeds the need of the normal tissue, the level of EPO in the circulation system is reduced. Because EPO plays a crucial role for the formation of red blood cells, this hormone has a broad potential for the treatment and diagnosis of blood diseases characterized by low generation of and/or defective red blood cells. Recent studies provide the basis for predicting the effect of EPO therapy in a variety of diseases, disorders, and hematological abnormalities. Applications of EPO include: the use of EPO in the treatment of anemia in patients with chronic renal failure (CRF), and administration of EPO to patients with AIDS and receiving chemotherapy (Danna, R. P., Rudnick, S. A., Abels, R I: edited by M B, Garnick, EPO in Clinical Applications—An International Perspective. Marcel Dekker; 1990: p 301-324).
Part of the biological effects of EPO can be regulated by interaction with surface receptors on the cell membrane. Previously, when studying the binding of EPO protein to cell surface using immature red blood cells isolated from mice spleen, it is found that this protein is composed of two polypeptides, having molecular weights of approximately 85,000˜100,000 KD (see Sawyer, et al. (1987) Proc. Natl. Acad. Sci. USA 84:3690-3694 for a more detailed description). The number of binding sites of EPO has also been calculated. Each cell membrane contains about 800 to 1000 sites. In these binding sites, about 300 binding sites have a Kd value of 90 pM. The binding of the remaining binding sites is weak, being about 570 pM. Some studies have shown that, from the response to EPO of red blood cells from the spleen of mice infected with the Friend virus anemia strain, about 400 binding sites are identified, wherein some have a high Kd of 100 pM and some have a low Kd of 800 pM.
Subsequent work has shown that the two types of EPO receptor are transcribed from a single gene. This gene has been cloned now. For example, the DNA sequences and peptide encoding sequences of the mouse and human EPO receptor have been described in WO90/08822. Current models show that binding of EPO to EPO receptor leads to activation and dimerization of two EPO receptors. This dimerization further leads to the initiation of signal transduction.
The cloned gene of EPO can be used to find agonists and antagonists of these important receptors. Peptides that interact to some extent with the EPO receptor have been identified and described. Specially, a group of peptides containing the major peptide fragment have been identified, which can bind to the EPO receptor and stimulate differentiation and proliferation of cells. However, the EC50 of the peptide that can stimulate differentiation and proliferation of cells is very low, ranging from 20 nM to 250 nM. Therefore, the clinical applications of these peptides are limited. The present invention provides EPO mimetic peptide derivatives, and their pharmaceutical salts, with biological activity and bioavailability, as well as methods for preparing the same.